The study was conducted at Muheza District Designated Hospital, in Muheza District, north-east Tanzania. Muheza district, (5°42' S and 38°48'E) has a human population of 278,405 and the inhabitants are mainly involved in subsistence agricultural production. The district is served by one district hospital, three health centres and 32 dispensaries. The lowland areas of the districts are areas of intense transmission 7. The entomologic inoculation rate in the area has been described as approximately 34 in the highlands and 400 infected mosquito bites each year in the lowlands 8.

The study population comprised of mothers aged between 18 and 45 years who had placental Plasmodium falciparum parasites and their respective neonates. Recruitment of the study participants was done from September 2002 to September 2005 from maternity ward at Muheza DDH and 1,022 mother-neonate pairs were screened. Mothers diagnosed with placental parasitaemia identified in a blood smear by microscopy and their respective neonates were enrolled in the study. Mothers with evidence of chronic or debilitating illness, such as recent significant weight loss, chronic diarrhoea, or history of chronic illness were excluded. All infants born from mothers diagnosed with placental parasitaemia were seen at birth and at two-week intervals for two years for full clinical examination and blood sampling, either at the Muheza Designated District Hospital or at the Projects sponsored mobile clinics that rotated through study villages on a weekly basis. Written informed consent was obtained from the mother prior to recruitment into the study.

Demographic information on maternal age, village of residence, gravidity, history of intermittent presumptive therapy (IPT) against malaria during pregnancy, abortion or miscarriage;as well as baby information on birthweight and gestation age were collected by trained nurses at enrollment. Bed net usage was assessed by village health workers during clinical visits and onsite home visits. The study protocol was approved by the International Clinical Studies Review Committee of the Division of Microbiology and Infectious Diseases at the US National Institutes of Health, and ethical clearance was obtained from the Institutional Review Boards of Seattle Biomedical Research Institute and the National Institute for Medical Research in Tanzania.

Blood samples of study mothers diagnosed positive for P. falciparum by microscopy were collected by the project staff nurses from placental tissues, umbilical cord and through either finger or heel prick of new born infants. Placental blood samples were obtained after delivery by mechanically grinding full thickness placental tissue 9. Cord blood was collected by cannulation of the thoroughly cleaned umbilical vessels while keeping the cord clamped. Blood samples from healthy infants were collected by heel prick every two weeks and at the time of any illness through mobile clinics until when they presented with first infection. In order to determine the outcome of placental malaria, all infants from mothers diagnosed with placental parasitaemia were followed for two years. All placental samples from these mothers tested P. falciparum positive in their thick smears and all infants born from these mothers who presented with parasitaemia within three months from birth were selected for further analysis by PCR. In order to analyse the phenomenon of malaria infections of neonates via transmission across the placental, matched umbilical cord blood samples were examined in thick blood smear and further analysed whether negative by Giemsa stain or not. About 10 μl of blood samples were bloated on 0.5 × 2 cm strips of 3 MM Whatman filter papers and dried in a laminar flow hood. After drying, the filter papers were packed in small individual plastic bags to avoid contamination and stored at room temperature until needed for analysis.

Genomic DNA was extracted from blood collected on 3 MM Whatmann filter papers using Generation^® capture card kit (Gentra Systems, Minneapolis, USA). The DNA was amplified in two nested PCR using oligonucleotide primers of the 5' and 3' conserved regions as described by 10. The details of two sets primer sequences used in the PCR for DNA amplification are shown in Table 1. PCR conditions included 94°C-1 min, 50.3°C-1 min, 72°C-45 sec., × 40, 72°C-5 min, 4°C-hold. In both reactions nest one and two PCR, reaction volumes ranged between 25 μl to 50 μl and the final concentration contained,5 mM Tris HCl (pH 8.3), 50 mM KCl, 0.5 mg of gelatin per ml, 2.5 mM MgCl₂, 200 μM mixture of dNTPs (Pharmacia Biotech, Uppsala, Sweden), and 0.25 U/μl of Taq polymerase (Boehringer, Mannheim, Germany). To each PCR tubes, 5 μl of DNA was added in primary reaction and 2 μl was re- amplified in the nested PCR reaction and carried out in a PTC^®-100 Peltier. The expected lengths of the bands were 509 bp and 750 bp and the previously established DNA positive control sample used was 3D7. In all PCR reactions a negative control sample with no template DNA was used.

Gel electrophoresis was performed on nested PCR products to confirm the presence of PCR amplicons size of interest. Placental-cord pair, placental-infants first parasitaemia and placental-cord and infants first parasitaemia samples were run in parallel in the 2% agarose gel in 100 ml of 1 × TBE (100 mM Tris, 100 mM borate and 5 nM EDTA) solution. The gels were stained with 0.5 μg/ml ethidium bromide final concentration and the similarities and differences of the bands size was visualized by ultraviolet light. Fragment sizes were estimated using a DNA size marker (10 μl of 100 kb ladder, GeneRuler™DNA ladders). Nest two PCR products with single bands were purified by QIAquick PCR product purification kit using microcentrifuge before sequencing. For samples which presented more than one band (more than one genotype), the gel were cut along those bands that share the size, purified by QIAquick Gel extraction kit and then sequenced.

DNA sequencing was done on nest two amplicons 11. In preparation for sequencing, excess dNTPs and unincorporated primers were eliminated from DNA by purification using QIAquick PCR purification kit (Qiagen). DNA sequencing was performed by fluorescence-based technique with an Applied Biosystems (Foster City, CA) 377 automated DNA sequencer. The purified PCR products, one prepared template were sequenced for each msp2 and the second template were sequenced if differences were found between the two derived sequences.

Data were analysed using STARTVIEW statistical package. Descriptive analysis used to evaluate the means of the different variables. Paired t- tests were used to analyse and determine significance relationship between difference observed variables. Regression plots that showed the variability around means of different variables and P value < 0.05 in the analysis was considered to be significant. Chi-square test was used to determine the proportional of the placental parasitaemia among gravity (primigravid, secundigravid and multigravid). A DNASTAR Program was used to analyse the sequencing results and all results were compared with msp2 sequences in Plasmodium genome resources or Plasmodium database (PlasmoDB) by Basic Logical Alignment Search Tool (BLAST) so as to determine if each individual sequence matched with concordant msp2 gene of P. falciparum sequences previously deposited.

By using PCR, placenta malaria was identified in 95 out of 100 microscopy-positive mothers and out of these 40 (42.1%) were primigravidae, 31 (34.7%) secundigravidae and 24 (25.2%) multigravidae. The mean weight of 95 babies born from placental malaria was 2.9kg with the range of 2–4.3 kg and 14.9% babies were below average birth weight while the mean gestation age was 36.3 weeks (range; 32–40 weeks). Seventy-six out of 95 (79.8%) infants were born before the normal gestation age. Infants born from mothers diagnosed with placental malaria during delivery, presented with the first clinical malaria at the mean age of 11 weeks (range; 8–64) weeks.

The prevalence of P. falciparum infection in placental, cord blood and infant's peripheral blood at first parasitaemia as diagnosed by microscopy and PCR are presented in Table 2. However, five (0.5%) of placental blood samples, which were positive by blood smear were negative by PCR regardless of repeat procedure. Twenty-three placental and five infant's peripheral blood sample had multiple infections (more than one strain of msp2 genes).

Fourteen pairs of placental and cord blood samples that shared the same band size following PCR were sequenced and analysed using DNASTAR program. Sequenced samples were paired based on similarities using Megalign program. Eight (60%) out of 14 pairs were genetically related while six pairs (40%) were genetically unrelated by the difference of few nucleotides estimated at 10%. In addition, seven pairs of infant's samples at first parasitaemia and corresponding placental samples shared same band size by PCR and were sequenced. Upon megalignment, one pair (14.3%) was genetically related by 100% while six pairs (85.7%) were genetically unrelated.

Comparisons of P. falciparum genotypes between and within pairs were also made. The comparison was between PCR results by looking at the bands which shared the same sizes, and sequencing results through alignment of the sequences in the phylogenetic tree. Some of the msp2 genes observed to be sharing the band size by PCR, had sequences not closely aligned in the tree. Nevertheless, pairs that were genetically related or closely genetically related by PCR were closely arranged in the tree leaves. Analysis from Plasmodium database (PlasmoDB) by Basic Logical Alignment Search Tool (BLAST) indicated that all samples originated from the msp2 gene of P. falciparum.

The prevalence of placental malaria which lead to symptomatic first parasitaemia significantly decreased as the gravidity increased (Regression coefficients, t = 4.8, P < 0.001) (Figure 1). Infants born from primigravidae were significantly more likely to be infected with P. falciparum (regression coefficients, t = 5.5, P < 0.001) as compared to infants born from multigravidae. It was observed that 8 out of 18 (44.4%) infants that presented with clinical malaria within three months after birth were from multigravidae mothers. Infants diagnosed with cord malaria by PCR were observed to be more susceptible to P. falciparum infection in their early life as compared to the infants that were cord negative, with the relative risk (RR) of 2.6 (Figure 2).